Trypanozoon subgenus has a unique sequence. As previously shown in blood from Guinean human African trypanosomiasis (HAT) patients, SL-RNA is an accurate target for diagnosis. Detection of SL-RNA in the cerebrospinal fluid (CSF) has never been attempted. In a large group of Congolese gambiense HAT patients, the present study aims i) to confirm the sensitivity of SL-RNA detection in the blood and; ii) to assess the diagnostic performance of SL-RNA compared to trypanosome detection in CSF.Spliced Leader (SL) trypanosome RNA is detectable only in the presence of live trypanosomes, is abundant and the p = 0.001). Of 96 CSF RNA specimens, 65 (67.7%) were SL-RNA positive, but there was no significant difference between sensitivity of SL-RNA and trypanosome detection in CSF. The contribution of DNA to the Cq values was negligible. In CSF with normal cell counts, a fraction of SL-RNA might have been lost during extraction as indicated by higher internal extraction control Cq values.Blood and CSF from 97 confirmed gambiense HAT patients from the Democratic Republic of Congo were collected using PAXgene blood RNA Tubes. Before RNA extraction, specimens were supplemented with internal extraction control RNA to monitor the extraction, which was performed with a PAXgene Blood RNA Kit. SL-RNA qPCR was carried out with and without reverse transcriptase to monitor DNA contamination. In blood, 92/97 (94.8%) HAT patients tested SL-RNA positive, which was significantly more than combined trypanosome detection in lymph and blood .This study was an integral part of the diagnostic trial "New Diagnostic Tools for Elimination of Sleeping Sickness and Clinical Trials: Early tests of Cure" (DiTECT-HAT-WP4, ClinicalTrials.gov Identifier:  Human African trypanosomiasis is a parasitic infection occurring in sub-Saharan Africa, which is fatal if left untreated. Diagnosis relies on demonstration of trypanosomes, which may occur at such low concentrations that they remain microscopically undetectable. Nucleic acid detection offers an alternative, in particular RNA, which is unstable and a better marker for live organisms than DNA. Trypanosomal SL-RNA detection in blood by reverse transcriptase quantitative PCR has hitherto only been tested twice. Although in cerebrospinal fluid, trypanosome presence indicates brain infection, SL-RNA detection has never been attempted. We evaluated sensitivity of SL-RNA detection in blood and cerebrospinal fluid. For each specimen, 2 controls were included: presence of genomic DNA contamination and efficacy of RNA extraction. Sensitivity of SL-RNA detection in blood was higher than of combined blood and lymph microscopy. In cerebrospinal fluid, SL-RNA and trypanosome detection had similar sensitivity. In a few specimens, traces of DNA were amplified. In some cerebrospinal fluids, some RNA was lost during extraction. Performing both internal controls is crucial, to ensure that negative SL-RNA cerebrospinal fluid findings are not due to a failed extraction and, in particular when testing treated patients, to differentiate live parasite RNA from reminiscent DNA. Trypanosoma brucei (T.b.) gambiense in Central and West Africa and by T.b. rhodesiense in East Africa. In humans, presence of the trypanosome in the blood and lymph only, corresponds to the first stage of the disease, whereas the second stage is characterized by trypanosome invasion of the central nervous system.Human African trypanosomiasis (HAT) or sleeping sickness is a neglected tropical disease caused by T.b. gambiense HAT, by the low and fluctuating trypanosomal load in these body fluids, intrinsic to the parasite and its interaction with the host’s immune response. The resulting low sensitivity of microscopic diagnosis, implies a risk of failure to detect the parasite although a patient is actually infected [The diagnosis of HAT is primarily based on the detection of the parasite in the blood, lymph or cerebrospinal fluid (CSF) . Performinfected .PCR targeting parasite DNA presents an alternative for the diagnosis of HAT . HoweverTrypanozoon subgenus has a unique sequence. SL-RNA is involved in the maturation of messenger RNA to which it is attached [T.b. gambiense, the specificity of SL-RNA detection in blood was higher than that of DNA detection by PCR [The detection of trypanosomal RNA, which is unstable and produced only by live parasites, demonstrates an active infection in the host and thus enables to establish a definite diagnosis. Spliced Leader (SL) trypanosome RNA can be used for diagnostic purposes, as it is abundant in trypanosomes and the attached . The diaattached ,6. Also n by PCR . To datend stage HAT, stage determination loses importance as new stage-independent treatments for sleeping sickness emerge [Although presence of trypanosomes in CSF has traditionally been a marker for 2s emerge –9. Nevers emerge . Also fos emerge ,12. The This study was an integral part of the diagnostic trial "New Diagnostic Tools for Elimination of Sleeping Sickness and Clinical Trials: Early tests of Cure" . The DiTECT-HAT-WP4 study has received approval from the “Comité Consultatif de Déontologie et d’Ethique” (CCDE) of the French National Institute for Sustainable Development Research , from the institutional Review Board of the Institute of Tropical Medicine (reference 1096/16), and from the Ethics Committees of the Ministry of Health, DRC through the Ngaliema Clinic of Kinshasa (September 2016 session) and the School of Public Health of the University of Kinshasa (reference ESP/CE/136/2020). It was carried out in conjunction with a prospective therapeutic trial conducted by DNDi  on the new drug Acoziborole for HAT that was approved by the CERSAC, Ethics Committees of the Ministry of Health, DRC through the Ngaliema Clinic of Kinshasa and the CNES , DRC.Participants first gave their informed consent to participate in the therapeutic trial. A separate written informed consent was obtained next before their inclusion in the DiTECT-HAT-WP4 study. For minors participating in the DiTECT-HAT-WP4 study, an additional written consent was obtained from their legal representative.The DiTECT-HAT-WP4 study was conducted in Democratic Republic of the Congo from February 2017 until March 2019. Patients were recruited in five reference district hospitals that were conducting the DNDi-OXA-02-HAT therapeutic trial under the supervision of the PNLTHA, located in Dipumba and Katanda in the province of Kasaï Oriental, Ngandajika in the province of Lomami, Masi Manimba in the province of Kwilu and in Kinshasa.T.b. gambiense parasites in blood, lymph and/or CSF; age ≥15 years; give and sign the study consent form; Karnofsky index > 50; able to take tablets orally; have a permanent address or being retrievable by other people; able to comply to the follow-up visits schedule and to other constraints of the study and accept to be hospitalized for acoziborole treatment and study procedures. As all HAT patients participating in DiTECT-HAT-WP4 were first enrolled in the DNDi-OXA-02 study, they were treated with acoziborole, independent of their disease stage.Patients included in the DITECT-HAT-WP4 study were enrolled first in the DNDi-OXA-02-HAT study. The inclusion criteria for DiTECT-HAT-WP4 corresponded to those applied in the DNDi-OXA-02-HAT study: confirmed HAT with T.b. gambiense test or a rapid diagnostic test for gambiense HAT). Individuals who were positive in a serological test underwent parasitological examination. For persons with swollen cervical lymph nodes, a lymph aspirate was examined for trypanosomes by microscopy. For persons positive in a serological test but negative in lymph or without swollen lymph nodes, blood was examined for trypanosome presence by mini anion-exchange centrifugation technique on buffy coat (mAECT-BC)[Patients included in this study were diagnosed either in the village by mobile teams (active screening), or at the reference district hospital (passive screening). Screening consisted of identifying characteristic clinical signs, in particular swollen cervical lymph nodes, and serological tests (CATT/mAECT-BC). If mAECmAECT-BC). The patwww.PreAnalytiX.com) according to the instructions of the manufacturer. After the collection of CSF for direct examination, the PAXgene Blood Tube was opened and 2.5 ml of CSF were collected by holding the tube under the lumbar puncture needle, after which the tube was closed. Both blood and CSF samples collected in PAXgene Blood RNA Tubes were inverted at least 10 times, and decanted immediately into 10 ml cryotubes. They were stored at -20°C for maximum 2 months before transport in liquid nitrogen to the HAT National Reference Laboratory at the Institut National de Recherche Biomédicale (INRB) in Kinshasa, where they were stored at -80°C until RNA extraction.From each HAT patient, 2.5 ml of blood were collected in a PAXgene Blood RNA Tube . The samples were thawed by placing them at ambient temperature for 4 hours to ensure full lysis before centrifugation and start of the extraction process. The pellet was lyzed and supplemented after centrifugation with 4 μl RNA internal extraction control (IEC) according to manufacturer’s recommendations . Briefly, the resuspended pellet was proteinase K treated, and residual cell debris removed through PAXgene shredder spin column centrifugation. Ethanol was added to the flow-through fraction and the lysate applied to a PAXgene RNA spin column centrifugation. The bound RNA was treated with DNase I and after several wash steps, RNA was eluted in 80 μl and stored at -80°C. RNA extracted from human blood spiked with T.b. gambiense LiTat 1.3 served as positive control.RNA extraction on blood and CSF samples collected in PAXgene Blood RNA Tubes was performed using a PAXgene Blood RNA Kit IVD following the guidelines of the manufacturer  with 400 nM of primers 5′-CAATATAGTACAGAAACTG-3′ (cSL) and 5′-AACTAACGCTATTATTAGAA-3′ (SL-F) (IDT Integrated DNA Technologies) and 4 μl RNA eluate in a final volume of 20 μl. All RNA extracts were tested in duplicate, and with (RT) or without (noRT) addition of reverse transcriptase (RT) . Cycling conditions using AriaMX Real-Time PCR System Machine  were 45°C for 10 minutes, 95°C for 2 minutes, and 40 cycles of 95°C for 2 seconds, 50°C for 10 seconds and 60°C for 5 seconds. The melting curve was recorded between 45°C and 95°C, using increments of 0.5°C for 5 seconds. Each qPCR run included a positive control from the spiked An RNA extract was considered positive for SL-RNA if both RT replicates were associated with a melting temperature (Tm) between 65 and 68°C. When detected, the Cq of the noRT reaction was used to calculate the percentage contribution of genomic DNA to the Cq-value of the RT reaction according to the following equation :%gDNA= and 5 μl of RNA extract in a 20 μl reaction. Cycling conditions using AriaMX Real-Time PCR System Machine  were 50°C for 10 minutes, 95°C for 2 minutes and 40 cycles of 95°C for 2 seconds, 50°C for 10 seconds and 60°C for 5 seconds.p value.For proportions, binomial confidence intervals with a 95% confidence level (CI) were calculated. The categorical results of trypanosome and SL-RNA detection in the same patients were compared by the Mc Nemar Chi square test. The Cq values of the IEC of two groups of patients were compared by an unpaired t-test, determining the two-tailed In total, 97 parasitologically confirmed HAT patients were included in DiTECT-HAT-WP4, 53 in Masi Manimba, 23 in Dipumba, 9 in Katanda, 10 in Ngandajika and 2 in Kinshasa. The median age of the participants was 31 years (interquartile range 24–44). The sex ratio was 50 females for 47 males. The CATT test was positive in 100% of patients on whom it was performed (95/95), the remaining two patients, on whom CATT was not performed, were positive in SD Bioline HAT. The parasitological results of these HAT patients are summarized in Swollen lymph nodes could be punctured in 53 patients, and lymph was trypanosome positive for 35/53 HAT patients included in the study . Next, blood was examined for 68 patients, and trypanosomes were detected in blood for 45 of 68 included HAT patients . Lymph or blood were trypanosome positive in 78/97 HAT patients . In total 69/97  HAT patients had trypanosomes in CSF, including all 19 patients in whom trypanosome infection could not be demonstrated previously in lymph and blood.Fourteen HAT patients were classified in early stage , 7/97 in intermediate stage  and 76/97 in late stage . All of them were SL-RNA positive, either in CSF and/or blood. Detailed results are shown in The internal extraction control added to each blood specimen during the RNA extraction procedure was detected by RT-qPCR in all 97 extracted blood specimens with a mean Cq value of 20.9 (standard deviation 1.0).Of 97 confirmed HAT patients, 92  tested SL-RNA positive in blood . The conp = 0.0001, 63 SL-RNA versus 45 parasite positives). SL-RNA detection in blood was also significantly more positive than combined lymph and blood parasite detection .Four out of 5 SL-RNA negative specimens  did not show amplification  and/or had a RT Cq value <40 but a melting temperature far outside the 65–68°C range. One negative specimen (patient 49) showed one positive reaction (Cq 33.6 and melting temperature 66°C) and one negative reaction . In only one of these blood SL-RNA negative patients, trypanosomes had been detected in blood and CSF (patient 58), while in the other 4 , trypanosomes had been detected in CSF only. Video recordings reconfirmed microscopic parasite finding for these 5 SL-RNA negatives. In the 68 patients in whom trypanosome presence in blood had been examined, SL-RNA was significantly more positive than trypanosome detection  and 7  were SL-RNA positive. Of 76 patients in late stage, 71  had SL-RNA positive results .Of 97 patients included, 96 CSF RNA specimens were available (CSF RNA specimen of patient 93 missing). The internal RNA extraction control added to each CSF specimen during the RNA extraction procedure was detected by RT-qPCR in all 96 specimens with a mean Cq corresponding to 25.4  were SL-RNA positive . Again, p = 0.5, 65 SL-RNA versus 68 modified single centrifugation positives).Of 14 early stage patients, zero  had SL-RNA in CSF, while of 7 patients in intermediate stage, one  reacted SL-RNA positive (patient 20). Of 75 CSF specimens from late stage patients, 64  contained SL-RNA. From 7 patients in late stage without trypanosomes in CSF, 2  were CSF SL-RNA positive (patients 22 and 27). From 68 late stage CSF samples containing trypanosomes, 62  were SL-RNA positive. In 6 patients who were SL-RNA negative but reported as modified single centrifugation positive , retrospective analysis of the video recordings reconfirmed presence of live trypanosomes in the CSF. There was no significant difference in the number of SL-RNA or trypanosome positives  are processed. A second control step introduced in the protocol was parallel qPCR analysis of the specimen without addition of reverse transcriptase. This enables the calculation of the contribution of genomic DNA to the obtained RNA result, thus guaranteeing that RNA was effectively detected instead of remnant DNA. Moreover, as it has been estimated that half-live of most trypanosomal mRNA is less than 100 minutes , the dettage HAT , which itage HAT . In the The present study also has some limitations. First, although we were able to demonstrate the high sensitivity of RNA detection in blood of HAT patients, we could not determine the specificity of the test, as no specimens from non-HAT control individuals had been collected. However, in 2 previous studies using a similar protocol in Guinea, specificities of respectively 96 and 100% were observed in 49 and 32 endemic healthy controls ,6. The sIn this study, the sensitivity of SL-RNA detection in the blood for diagnosis of HAT was 95.8% (CI 89.8–98.8%). In the past, the value of this parameter was determined at 92% (CI 78–97%)  and alsoDetection of trypanosomes in CSF is a marker for the diagnosis of second stage HAT . Howevera posteriori confirmation of HAT in serological suspects who undergo treatment without parasitological examination, as could be the case in future “test and treat” algorithms [The high sensitivity of SL-RNA RT-qPCR opens the door for future diagnostic applications. However, as for PCR in the past, SL-RNA is, for the moment, not really suitable for routine diagnosis of HAT under field conditions in rural areas where HAT occurs. It requires demanding and critical logistics, including correct specimen collection on RNA stabilization buffer, and transport of the stabilized RNA soon after collection or frozen from the HAT treatment center to a reference lab for proper RNA extraction, which cannot be expected in routine . In addigorithms .T.b. gambiense should be explored further. In particular the high sensitivity of RNA detection in blood could open the door for less invasive diagnosis of relapses, avoiding lumbar puncture. Furthermore, as recently new RNA detection tests have emerged which have only been tested experimentally, including 7SL-derived small RNA [Future research should confirm specificity of RNA detection and confirm its sensitivity in CSF. The performance of detection of SL-RNA as a marker for the diagnosis of relapses during post-treatment follow-up of patients with mall RNA , future In conclusion, with the present study, we confirmed the elevated sensitivity of SL-RNA detection in the blood of HAT patients and demonstrated the diagnostic performance of SL-RNA detection in CSF for the first time, which is a first step towards new applications of RNA detection.


The sensitivity and specificity for differentiating between severe and mild influenza using our combined method  were 78.3 and 50.0%, respectively. When interleukin-6 was combined with serum C-reaction protein, the sensitivity and specificity were 85.7 and 95.5%, and the receiver operating characteristic area-under-the-curve was quite high . The potential advantages of our system, i.e., a paper-based test strip coupled with a spectrum-based optical reader, are as follows: 1) simple user operation; 2) rapid turnaround times–within 20 min; 3) high detection performance; and, 4) low-cost fabrication.Influenza virus infection is a major worldwide public health problem. Influenza virus infections are associated with a high hospitalization rate in children between the ages of 5 and 14. The predominant reason for poor influenza prognosis is the lack of any effective means for early diagnosis. Early diagnosis of severe illness is critical to improving patient outcome, and could be especially useful in areas with limited medical resources. Accurate, inexpensive, and easy-to-use diagnostic tools could improve early diagnosis and patient outcome, and reduce overall healthcare costs. We developed an interleukin-6 paper-based test strip that used colloidal gold-conjugated antibodies to detect human interleukin-6 protein. These complexes were captured on a paper-based test strip patterned with perpendicular T lines that were pre-coated with anti-human interleukin-6 antibodies. Applied serum samples interacted with these antibodies and presented as colored bands that could be read using a spectrum-based optical reader. The full-spectrum of the reflected light interleukin-6 protein signal could be obtained from the spectral optics module, and the standard could be used to quantitatively analyze interleukin 6 level in serum. We retrospectively evaluated 10 children (23 serum samples) with severe influenza virus infections, 26 children (26 serum samples) with mild influenza virus infections, and 10 healthy children (10 serum samples). Our system, the combined use of a paper-based test strip and a spectrum-based optical reader, provided both qualitative and quantitative information. When used with the optical reader, the detection limit was improved from a qualitative, naked-eye level of 400 pg/ml to a quantitative, optical reader level of 76.85 pg/ml. After monitoring serum interleukin-6 level via our system, we found a high correlation between our system results and those obtainable using a conventional sandwich enzyme-linked immunosorbent assay method (Rho = 0.706,  Influenza virus infection is a major worldwide public health problem. Seasonal infections with the common influenza virus strains  are frequently resolved, but still cause high mortality. The annual morbidity rate caused by influenza is the highest in children, where morbidity is usually >30% . ChildreInflammation is a rapid and non-specific host defense mechanism that is strictly regulated by a network of inflammatory mediators. Inflammatory mediators include cytokines, such as interleukin-6 (IL-6) . IL-6 isAlthough conventional methods to gain information via the use of biomarkers are common, most examinations must be performed in hospitals and require time, expensive/complex equipment, and well-trained professionals to facilitate inspection and analysis. Based on current global medical trends, we predict that the future medical ecosystem will provide an increasing number of opportunities for improvement via the use of point-of-care (POC) tools at the bench and at bedside . Rapid dHere, we discuss the design, prototyping, and testing of a paper-based test strip that can detect IL-6 via lateral flow immunoassay (LFA), and can be qualitatively read with the naked eye (detection limit of 400 pg/ml). To increase the detection limit of the test strip, a small, light, and easy-to-use spectrum-based optical reader, a collaborative effort with SpectroChip Inc. in Taiwan, was integrated into the methodology in order to capture the reflection spectrum on the test strip for analysis. This research and our perspectives could provide options and impetus for the development of a POC device for influenza diagnosis with an eye toward easy, early diagnosis as a means of improving patient outcome. Our results may also be useful for the development and implementation of methods for determining influenza severity and optimizing treatment, which would be especially useful if applied to a pediatric population.Mycoplasma pneumoniae infections, were only used for correlation analysis between our IL-6 test strip protocol and ELISA methodology . The protocol for this study was reviewed and approved by the Institutional Review Board (IRB) of National Cheng Kung University Hospital (IRB No: B-ER-102-345). Informed consent was obtained from each participating patient or the patient’s parents or guardian.Patients with virologically confirmed influenza infection visiting National Cheng Kung University Hospital, Taiwan were recruited. Influenza infection was defined as having clinical presentation of acute respiratory disease, plus virological evidence of influenza infection, either through positive influenza viral antigen test  or influenza virus isolated from respiratory specimens. The influenza infection cases were then further classified into mild or severe cases. Mild cases included those with only fever and respiratory symptoms, and severe cases presented lower respiratory tract infection, acute respiratory distress, hemodynamic instability, or severe extrapulmonary manifestation . Children without fever, respiratory symptoms or any sign of acute infection within 1 week were enrolled as healthy controls. Serum samples from another infectious disease cohort, including patients with dengue virus, enterovirus, and Soluble IL-6 concentration in serum samples was determined using monoclonal antibody enzyme-linked immunosorbent assay (ELISA) kits . The minimum detectable concentration was 3.1 pg/ml. The intra- and interassay coefficients were less than 7%. The ELISA reader brand was Tecan (SunriseTM | Absorbance Microplate Reader). The experiment protocol and analyzing method all follow the standard protocol of the ELISA kit. We measured IL-6 concentration in all 83 serum samples.This paper test strip (created in cooperation with Taiwan Hygeia Touch Inc.) used colloidal gold-conjugated anti-IL-6 antibody  to detect human IL-6 antigen. Based on the colorimetric analysis of each test combination, an optimization test was performed to determine the most suitable IL-6 concentration and gold nanoparticle size for conjugation. The test strip was pre-coated with capture reagent and sprayed to generate vertical lines containing anti-IL-6 antibody  bound to 15 nm-diameter gold colloid. The C line of the nitrocellulose strips was also coated with mouse anti-human IgG antibody  and an equal amount of quality control antibody. When the applied sample flowed along the strip, the human IL-6 antigen present in the sample formed an antigen complexed with the colloidal gold-labeled anti-IL-6 antibody. These complexes are then captured in the vertical T-line of the test strip, where they appeared as colored strips.The spectrum analyzer -008090 and US FDA: 3017810861) was equipped with a cassette tape designed to accept the paper-based test strip and could detect the IL-6 antigen reflectance spectrum from the test paper. This device provided continuous spectrum results and captured high-resolution reflectance spectrum values from the test strip test line (T line). The spectrum reader provided high resolution (3–5 nm) results over a wide spectral range . The principle of machine detection is to scan with white light. When the red line was scanned, the colloidal gold absorbed the spectrum between 430 and 600 nm and reflected the remaining light that was not absorbed by the colloidal gold. Colloidal gold does not absorb 650 nm light, so the red lines of different shades produced little change at 650 nm, which is approximately equal to 1.The main reflection wavelengths detected by this spectrum reader were 430 and 600 nm (for samples with lower IL-6 concentration), and the main reference wavelength was 650 nm. The ratio of the minimum reflectance to the spectrum at the reference wavelength was used to calculate the α value:In this formula, α refers to the color reflection value of the optical scanning IL-6 antigen rapid screening test strip. The higher the α value, the stronger the reflection color intensity of the IL-6 complex coupled with the colloidal gold antibody, which indicated higher the IL-6 concentration.The LOD and LOQ were estimated based on the average of the blank α value, the standard deviation of the blank α value, the slope of the calibration curve  and the defined confidence factor, using the following calculations:In this calculation, the average blank α value was 1.00395, the standard deviation of the blank α value was 0.00278, the blank coefficient of variation was 0.27%, and the 95% confidence interval was 99.6–100%. LOD was estimated by using the average value of blank α value plus 3 times the standard deviation of the average value of blank α value, using the following equation in p-value less than 0.05 was considered statistically significant. The receiver operating characteristic (ROC) area under the curve (AUC) were used to evaluate the diagnostic ability of IL-6. In order to evaluate the correlation between this paper-based test strip/optical reader combination and ELISA, we used Spearman’s rank correlation coefficient and the Bland-Altman chart.The Wilcoxon rank sum test and the Wilcoxon signed rank test were used to check data and distinguish severe cases, mild cases, and healthy controls. A A total of 36 influenza infected patients  and 10 healthy individuals (10 serum samples) were recruited. The clinical characteristics and investigative findings are provided in As shown in R2 value of 0.9507. Based on this regression model, the LOD and limit of quantification (LOQ) were 76.85 pg/ml and 402.71 pg/ml, respectively. Based on these results, the platform provides an excellent analytical sensitivity  for the detection of trace levels of human IL-6 protein.The percentage of the reflectance spectrum is negatively correlated with the number of antibody-antigen complexes, so it decreases as IL-6 concentration increased. Here, we found that the reflectance spectrum of IL-6 was well separated at about 540 nm . The refp < 0.001).We selected 83 patients, all of which were children that had different illness severities. We then measured each patient’s serum IL-6 concentration using two tests: 1) the IL-6 test strip coupled with a spectrum-based optical reader; and, 2) enzyme-linked immunosorbent assay (ELISA). As shown in p = 0.46)  between the test strip system and ELISA. The Bland-Altman diagram simply represents the comparison of each difference between the two pairing methods with the measured average, as shown in  = 0.46) . The meap = 0.071). The IL-6 level results provided by the conventional sandwich ELISA method (p < 0.05). Although there was no significant difference in IL-6 level between severe and mild influenza cases, two cases with serial serum samples demonstrated that IL-6 level correlated with disease progression , respectively. The cut-off value according to the test strip ROC curve was 140.4 pg/ml, and the sensitivity and specificity were 78.26 and 50.0% (p < 0.05 and p > 0.05). Combining IL-6 with CRP provided a high AUC in the ROC curve for the test strip results. The sensitivity and specificity for IL-6, when combined with CRP, were 85.71 and 95.45%; the sensitivity and specificity for IL-6, when combined with CRP, were 71.43 and 63.64%. Of the other available clinical information, i.e., the number of days in the hospital, the number of days in the ICU, and highest body temperature, the first two values  correlated with test-strip IL-6 concentration  (The ROC curve could be useful for summarizing the performance of each classifier into a single measure. It shows the trade-off between sensitivity (or TPR) and specificity (1–FPR). As shown in nd 50.0% . We calcnd 50.0% , such as < 0.01) .In order to provide a rapid, simple, precise, and inexpensive device, we designed an easy-to-use POC system. As shown in More than half (55%) of all children hospitalized for influenza have underlying respiratory diseases, including asthma (27%) . When a In this study, compared with mildly affected patients, severely affected patients had higher IL-6 concentrations, but the difference was not statistically significant. There may be some other factors we need to discuss, for example, the number of days that a patient had a fever before admission might affect the process of the disease, the immune system of children is not mature, and there are different type of influenza, etc.  In the fIL-6 may be considered a potentially useful biomarker for influenza testing, and its significance as a biomarker may be further underscored by its associated rise in patients with encephalopathy . To dateTo conclude, the advantages of our system, i.e., a paper-based analytical device coupled with a spectrum-based optical reader, are as follows: 1) simple user operation; 2) rapid turnaround times–within 20 min; 3) high detection performance; 4) low-cost fabrication; and, 5) small size, which saves workbench space and facilitates use in any medical environment. In addition, our system may contribute the following to the medical arena: 1) promotion of effective patient management; 2) assistance in targeted antiviral therapy and antibiotic management; 3) facilitation of immediate infection control measures; and, 4) reliable early-stage disease detection that could reduce overall medical costs .


Cracked teeth may cause various clinical symptoms depending on the extension depth of the crack and the subsequent bacterial infections. However, techniques to reliably determine the extension depths of cracks in teeth before treatment are lacking. The aim of this study was to develop a new technique based on contrast-enhanced cone beam computed tomography (CBCT) to improve the accuracy of crack depth evaluation in vitro.We developed an in vitro artificial simulation model of cracked teeth. Pre-experimental CBCT (pre-CBCT), and micro-computed tomography (micro-CT) were first performed for all cracked teeth (n = 31). Contrast-enhanced CBCT was then performed by infiltrating the crack with ioversol under vacuum conditions. The sensitivities of pre-CBCT and contrast-enhanced CBCT for the diagnosis of cracked teeth were calculated. According to the K-means clusters, crack depths measured by micro-CT were changed into categorical variables. Bland–Altman plot and the intraclass correlation coefficient (ICC) were used to analyze the consistency of the crack depths between the pre-CBCT and contrast-enhanced CBCT, as well as the ICC between the contrast-enhanced CBCT and micro-CT. Receiver operating characteristic (ROC) curves were generated to assess the ability for predicting crack depth in the differential diagnosis using pre-CBCT and contrast-enhanced CBCT. Restricted cubic splines were also used to model the non-linear relationship between the crack depths of contrast-enhanced CBCT and micro-CT.P < 0.001). The areas under ROC curves (AUC) of pre-CBCT and contrast-enhanced CBCT were different: the AUC of pre-CBCT was 0.958 , and the AUC of contrast-enhanced CBCT was 0.979 , and the difference was not statistically significant . The ICC value of crack depth as measured by contrast-enhanced CBCT and micro-CT was 0.753 .The sensitivities of pre-CBCT and contrast-enhanced CBCT were 48.4%, and 67.7%, respectively. The ICC value of crack depth as measured by pre-CBCT and contrast-enhanced CBCT was 0.847 (95% confidence interval [CI] 0.380–0.960; Contrast-enhanced CBCT under vacuum conditions with a contrast medium can significantly improve the crack detection rate of cracked teeth; however, it cannot measure the crack depths accurately. Tooth cracks extend from the crown toward the apex and gradually propagate into the dentin, involving one or two marginal ridges , 2. CracSeveral auxiliary approaches for diagnosing cracked teeth exist, including operating microscopes , 8, tranSeveral studies have investigated the diagnosis and depth assessment of cracked teeth in vitro. Imai et al. –19 reporRadiological examination, such as cone beam computed tomography (CBCT), is a commonly used diagnostic tool. However, it can only be used to diagnose wide cracks , 24. AddIn this study, to improve the effectiveness of CBCT as a tool for determining the crack depth of cracked teeth before treatment, we aimed to explore a technique based on contrast-enhanced CBCT, and investigated whether it would predict the crack depth of cracked teeth.From January to December 2018, we collected 200 intact teeth extracted using minimally invasive techniques for periodontal or orthodontic reasons at the Department of Oral and Maxillofacial Surgery, Stomatological Hospital of Tianjin Medical University. This study was approved by the ethics committee of the Stomatological Hospital of Tianjin Medical University (TMUhMEC2019014). The inclusion criterion was the absence of enamel cracks that were visible to the naked eye. The exclusion criteria were the presence of dental caries, wedge-shaped defects, and VRFs, and a previous history of root canal therapy or restoration. Finally, 40 teeth that met the criteria were screened in vitro.An in vitro artificial simulation cracked tooth model was established. All the 40 screened intact teeth were cleaned to remove soft tissue and calculus, and soaked in 0.1% thymol solution for 24 h. Subsequently, they were placed in 0.9% saline solution, soaked in liquid nitrogen (− 196 °C) for 1 min and immediately transferred into hot water (90 °C) for 5 min , 33. TheThe pre-CBCT  parameters were as follows: voltage, 120 kV; current, 8 mA; matrix size, 640 × 640; field of view, 8 × 8 cm; and pixel size, 0.125 mm × 0.125 mm. To ensure better pre-CBCT imaging quality, the operations were performed by a technician. The pre-CBCT results were stored in a database, and pre-CBCT images were analyzed using the Vision Q (KaVo eXam Vision) software package.The micro-CT  parameters were as follows: voltage, 80 kV; current, 500 μA; resolution, 9.08 μm; exposure time, 1000 ms. The Inveon Research Workplace software  was used to reconstruct three-dimensional (3D) images. The presence of cracks and their extension depths, as measured from the micro-CT images, was recorded by a radiologist who was blinded to the evaluation of the pre-CBCT images.The procedure to measure crack depth extending from the crown to the root on micro-CT images Fig. A–C was a18H24I3N3O9; Hengrui Pharmaceutical, Jiangsu, China) was diluted in a ratio of 3:1 in normal saline to increase its fluidity. The crown and root of each cracked tooth were isolated with a rubber dam, and the crown was filled with the diluted ioversol solution. The teeth and the rubber dam were kept in place using a rubber band and a paper cup  probably or definitely not a lesion, (ii) probably or definitely a lesion.On contrast-enhanced CBCT, a high-density linear crack was considered to mark a cracked tooth. The method used for measuring cracks on contrast-enhanced CBCT was the same as that used for micro-CT: the position of the tooth was adjusted in the sagittal and coronal planes to set it upright; the coronal plane of the crack shows its path Fig. A; the crhttp://www.r-project.org/), and RStudio 1.1.463  were used to perform the statistical analysis for the study.SPSS v.26.0 software , R software, version 3.6.0  was used to evaluate the consistency of the crack depths as measured on pre-CBCT and contrast-enhanced CBCT, as well as between contrast-enhanced CBCT and micro-CT. The cracked teeth that could be detected by pre-CBCT and contrast-enhanced CBCT were included in the receiver operating characteristic (ROC) analysis. The crack depth of pre-CBCT and contrast-enhanced CBCT were continuous variables, and the crack depth of micro-CT was changed into a binary variable to study the predictive ability of pre-CBCT and contrast-enhanced CBCT for crack depth.Moreover, we used restricted cubic splines (RCS) with 4 knots at the 5th, 35th, 65th, and 95th percentiles to model the non-linear relationship between the crack depths of contrast-enhanced CBCT and micro-CT.The results of the pre-CBCT, contrast-enhanced CBCT, and micro-CT were recorded Fig. A–C. ThirThe sensitivity values of pre-CBCT and contrast-enhanced CBCT for diagnosing cracked teeth were 48.4%, and 67.7%, respectively, implying that the sensitivity of contrast-enhanced CBCT was higher than that of pre-CBCT Fig. B.The mean crack depth of the 10 teeth that were only measured on contrast-enhanced CBCT, but not visible on pre-CBCT was 7.114 ± 1.587 mm. Meanwhile, the mean crack depth of the 4 cracked teeth detected only during pre-CBCT, and not during contrast-enhanced CBCT, was 7.2650 ± 1.135 mm.P < 0.001), demonstrating strong consistency. Both pre-CBCT and contrast-enhanced CBCT exhibited good consistency in determining the crack depth  of pre-CBCT was 0.958 , and that of contrast-enhanced CBCT was 0.979 . The AUCs of pre- and contrast-enhanced CBCT were different; however, the difference was not statistically significant , demonstrating strong consistency. Contrast-enhanced CBCT can explain the 72.36% of micro-CT changes, and the root mean square error was only 1.81. We observed a linear relationship between the crack depths measured by contrast-enhanced CBCT and micro-CT  the crack direction of the artificial model in this study is uncertain, making ioversol infiltration difficult; (ii) the crack depth in the present study was not controlled, and the above two sets of data are not comparable.Our study revealed no significant differences between pre-CBCT and contrast-enhanced CBCT in measuring the crack depth of cracked teeth. This may be because crack width impacts the diagnostic accuracy of CBCT in detecting cracked teeth –43. If tFurthermore, the crack depths measured on contrast-enhanced CBCT and micro-CT demonstrated good consistency. The result of RCS revealed that the relationship between the difference in the crack depths measured on contrast-enhanced CBCT and micro-CT to be linear. However, a formula cannot be obtained to calculate the specific relationship between the depth measured by contrast-enhanced CBCT and micro-CT.It is worth mentioning that the crack depth measured by both micro-CT and contrast-enhanced CBCT was a vertical distance from the crown to the root, i.e., it was not the actual length of the crack. Considering that the crack line passing through the three different dimensions may exhibit inconsistencies and that most cracks were oblique, it was difficult to access the actual full length of the crack in a single plane. Clinically, the prognosis for teeth with cracks that extend vertically through the pulp, or involve the subpulpal floor, is less favorable . TherefoThere are some limitations to our study: (i) the sample size was small. Thus, the results need to be further confirmed using a larger sample size; (ii) the study was conducted in vitro; thus, the feasibility of the clinical application of this technique needs to be confirmed in the future; and (iii) the diagnostic capacity for the crack-width axis of the infiltrated contrast agent should be analyzed in future studies.We conducted this study and aimed to develop a new technique based on CBCT to improve the detection of cracked teeth and the accuracy of crack depth evaluation in vitro. The study suggests that CBCT with a high-density contrast medium is reliable for detecting cracks. On the basis of our findings, simulated animal experiments in controlled negative pressure conditions are warranted. Moreover, if this method is viable, we might have resolved the challenges faced while managing cracked teeth.Contrast-enhanced CBCT under vacuum conditions with a contrast medium can significantly improve the crack detection rate of cracked teeth; however, it cannot measure the crack depth accurately.


IntroductionLung cancer is the most common cancer overall, and the foremost cause of cancer-related mortality. Almost all lung cancers evolve from pulmonary nodules. As multidetector CT (MDCT) scanners are now widely available, there is an increased rate of detection of pulmonary nodules. It is of utmost importance to evaluate pulmonary nodules to rule out the possibility of neoplastic diseases. With advancements in technology, there are various manual and automatic analytic software providing a wide range of post-processing techniques. Maximum intensity projection (MIP) and volume rendering (VR) techniques have been analyzed previously regarding pulmonary nodules but there is a scarcity of data in terms of low-density nodules. This study aims to delineate the comparison and supremacy of both techniques in terms of low-density nodules.MethodologyThe current prospective study was conducted from June 2019 to June 2020 in the Radiology Department at Dr. Ziauddin Hospital, Karachi. Chest CT scans were performed on 16 slice MDCT . A consultant radiologist of six years experience and a postgraduate trainee of three years experience analyzed each patient on a workstation . SPSS 23.0  was incorporated for data analysis. Data were expressed in the median and interquartile range (IQR). Data collected for this study were analyzed using analyzing the median difference in nodule count using Wilcoxon’s signed-rank test. A p-value of <0.05 was considered significant.ResultsAfter informed consent, 236 patients were recruited for the study. MIP outperformed VR in terms of nodule detection and low-density nodules at each evaluated slab thicknesses (p<0.001). A 10-mm MIP was superior to all other techniques in terms of detection of pulmonary nodules and low-density nodules (p<0.001). MIP was also considered an easier technique as there was excellent inter-rater reliability and agreement.ConclusionThis study is robust evidence regarding the supremacy of MIP. MIP outperformed VR on every slab thicknesses. The 10-mm MIP technique was superior to all others evaluated and was recorded to be an easier analyzing technique. According to World Health Organization, lung cancer is among the most frequent cancer overall and is the noteworthy cause of cancer-related mortality ,2. The eIn MIP, voxels having the highest attenuation are projected along lines through the volume of a given dataset . In contWe evaluated MIP and VR on different slab thicknesses for detecting pulmonary nodules. Our objective of the study was to compare MIP and VR on slab thicknesses of 4-mm, 7-mm, and 10-mm in detecting low-density pulmonary nodules. To the best of our knowledge, very fewer studies have been performed in the past.Research designThis prospective study was conducted from June 2019 to June 2020 in the Radiology Department at Dr. Ziauddin Hospital, Karachi. A favorable ethical approval from the Institutional Research Review Committee was obtained. The study participants were provided a patient information sheet. After written informed consent, all patients of age more than 18 years were consecutively sampled who were referred to our institute for chest CT scan, irrespective of gender. By keeping the sensitivity of 86%, the specificity of 82%, with a 20% prevalence of pulmonary nodules and keeping precision at 10% using an online sample size calculator for sensitivity and specificity studies, a sample size of 236 was estimated which was sufficient for both sensitivity and specificity calculation in our study. Datasets with breathing artefacts, consolidation, pulmonary fibrosis, nodules count >20 in each lung, and pneumoconiosis were excluded from the study.Image acquisitionA 16-slice multidetector CT scanner  was used for scanning the chest in the craniocaudal axis from the apices of the lung to the upper abdomen. Scanned data were obtained in helical mode. The acquisition parameters were: tube voltage, 90-135 ㎸ (adjusted according to weight of the patient); collimation, 16 × 1; gantry rotation, 0.75 sec; pitch, 1.0; slice thickness, 1-mm; reconstruction kernel, FC 50-52; and reconstruction increment, 0.6-mm. The required output of tube current-time product was adjusted automatically according to the patient by in-built Toshiba’s Adaptive Iterative Dose Reduction (AIDR) technology. No intravenous contrast was used. Appropriate instructions were conveyed to the patient for breath-holding to minimize breathing artefacts.Image analysisImages were analyzed using image viewing software  through a cinematic viewing approach. Data for different characteristics of pulmonary nodules were recorded. Each scan was interpreted by two radiologists independently, one having experience of six years and a postgraduate trainee of three years’ experience, respectively. The software was used to reconstruct MIP and VR images. Window level of −400 HU and window width of 1600 HU were set for analysis of lung nodules on MIP technique, while VR images were evaluated at −500 HU window level and width of −1500 HU. Opacity for VR images was kept at 50%. Both radiologists analyzed each scan six times using the following method: MIP and VR on slab thicknesses of 4-, 7-, and 10-mm, respectively. Larger slab thicknesses were not used because of overlapping of structures and partial volume artefact. Although the analysis was done in cinematic axial approach at times, doubtful nodules were confirmed after seeing the multiple planes. Nodules characteristics such as size and density were evaluated on both post-processing techniques (MIP and VR). Classification of nodules was done on sizes of <6-mm and >6-mm. The density of nodules was categorized as low if HU ≤ 100 and high if HU > 100. Both readers were unaware of the number of pulmonary nodules in each patient. MIP and VR images were analyzed separately with a minimum gap of at least one week to reduce memory effects.Data analysisStatistical analysis was performed using SPSS (IBM SPSS Statistics 23.0). Data were expressed in the median and interquartile range (IQR). Data collected for this study were analyzed using analyzing the median difference in nodule count using Wilcoxon’s signed-rank test. A p-value of <0.05 was considered significant. Intraclass correlation coefficient estimates (for interrater reliability) and their 95% confidence intervals were calculated using SPSS statistical package version 23  based on a mean-rating (k = 3), absolute-agreement, two-way mixed-effects model. The agreement was classified using a grading system: values down from 0.5 indicate poor reliability, values ranging from 0.5 to 0.75 stipulate moderate reliability, values range 0.75-0.9 stipulate good reliability, and values exceeding 0.90 stipulate excellent reliability .Two hundred and thirty six patients CT were examined; using MIP technique, more pulmonary nodules were observed  as compared to VR technique , respectively. Low density nodules were also detected more on MIP  than on VR .Interclass correlation coefficient for the calculation of 4-mm MIP was 0.99  0.99  for 7-mm MIP, and 0.99  for 10-mm MIP which indicated excellent inter-rater reliability and agreement. While, interclass correlation coefficient for the calculation of 4-mm VR was 0.81 , 0.89  for 7-mm VR, and 0.91  for 10-mm VR which indicated good to excellent inter-rater reliability and agreement.As the availability of MDCT has widely grown, the detection of pulmonary nodules has increased. Post-processing software provides various reading techniques such as average, MIP, MinIP, and VR. Recent technical advancements based on artificial intelligence, i.e., computer-aided detection have also shown promising results . HoweverWe found that number of nodules were detected more on the MIP technique than VR technique for each of the observer, and there was a statistically significant difference between these two techniques at every slab thickness, p-value < 0.001 (Table The size was another criteria, we evaluated supremacy of these two techniques in only two categories, i.e., <6-mm and >6-mm, as there are plentiful data in the previous studies -8. SharmWe also found that using MIP enhanced detection of low-density nodules, despite the fact that anatomic details are preserved by VR technique . In our Although we did not evaluate reading time for each of the datasets, however, we found that both users were able to report MIP in a lesser time duration than VR, which represents MIP being a user-friendly technique.There were few limitations of the study. We postulated results on the data recorded by reader A; the other reader was resident for which we evaluated inter-rater agreement only. The excellent inter-rater agreement shows that MIP is easier to interpret and is adjustable according to the reader’s professional experience. Our study also lacks a true reference standard because histopathological proof cannot be obtained, and we do know that only a CT scan can provide nodule characteristics in a non-invasive approach. As the data remains scarce, more work is required to evaluate the role of MIP and VR in the evaluation of low-density nodules.This study is robust evidence that the MIP technique is superior to the VR technique in terms of nodule detection as well as detection of low-density nodules. In our study, MIP outperformed VR on each analyzed slab thickness. MIP was also easier to interpret and had an excellent inter-reader agreement. The 10-mm MIP was superior for nodule detection as well as low-density nodules over all the analyzed slab thicknesses.


Purpose: To assess the outcomes of implanting a new polymethylmethacrylate (PMMA) ring  in pre-descemet deep anterior lamellar keratoplasty (PD-DALK) procedure for moderate-advanced keratoconus.Methods: This prospective study included 10 eyes of 10 patients with moderate-advanced keratoconus who underwent PD-DALK with Neoring implantation. Neoring was implanted in a pre-descemetic pocket. The post-operative examination included refraction, corrected distance visual acuity (CDVA), corneal tomography, and endothelial cell density (ECD). The root mean squares (RMSs) for coma-like aberrations and spherical aberration were evaluated for a pupil size of 4.5 mm. The junctional graft (Tg) and host (Th) thicknesses were measured. The post-operative follow-up was 24 months.Results: Post-operative CDVA was 0.82 ± 0.14 , 100% of the eyes achieved a CDVA of 0.7 . The refractive cylinder was −2.86 ± 1.65 2-years after surgery. No eyes had a post-operative refractive cylinder ≥5.00 D and in five eyes (50%), it was ≤2.50 D. At the last visit, the mean keratometry was 45.64 ± 1.96 D, the RMS for coma-like aberrations was 0.30 ± 0.15 μm and spherical aberration was 0.22 ± 0.09. The mean ECD remains without changes over the follow-up (P = 0.07). At the last visit, Tg and Th were 679.9 ± 39.0 and 634.8 ± 41.2 μm, respectively. The thickness of the complex (host-Neoring) was 740.6 ± 35.6 μm. In all cases, this thickness was thicker than Tg.Conclusion: The results of this study suggest that PD-DALK along Neoring implantation is a viable, effective, and safe option to optimize the post-operative results for moderate-severe keratoconus. Deep anterior lamellar keratoplasty (DALK) is a surgical procedure in which a corneal donor, free from endothelium and Descemet's membrane (DM), is transplanted in patients affected by a corneal disease, in which the endothelium is healthy and can be preserved. DALK may be classified into two types according to the technique used: with and without DM exposure, known as Descemet-DALK (D-DALK), and pre-descemet DALK (PD-DALK), respectively. Overall, DALK has significant advantages over penetrating keratoplasty (PKP), including reduced risk of endophthalmitis, no immune reaction, and minor loss of endothelial cell density (ECD), among others . HoweverThe ocular disease leading to corneal transplantation might also influence the DALK-related complications , 9–11. AWe designed a new ring made of polymethylmethacrylate (PMMA), named Neoring , to overcome the potential complications of DALK in advanced keratoconus. First, the Neoring is conceived for the PD-DALK technique owing to the fact that it is implanted in a PD pocket created through the trephination periphery. As previously pointed out, the most common intraoperative complication (rupture DM) is mainly associated with D-DALK. Second, the Neoring implantation aims to regularize the graft-host interface and recipient corneal bed. Notedly, that corneal graft conforms, in part, to the recipient bed. Hence, the Neoring implantation might lead to a better corneal morphology restoration, consequently, improve the visual and refractive outcomes after surgery.In the current prospective study, 10 eyes of 10 patients who underwent PD-DALK with Neoring implantation for advanced keratoconus were case per case analyzed over a follow-up of at least 24 months.This is a prospective case series study that included patients with moderate-advanced keratoconus, who underwent PD-DALK surgery with Neoring implantation at the Fernández-Vega Ophthalmological Institute in Oviedo, Spain. This study was conducted in compliance with the tenets of the Declaration of Helsinki. The study was approved by the Ethics Committee of the “Principado de Asturias” . After receiving a complete description of the nature of the study and the possible consequences of surgery, all patients provided informed consent.Inclusion criteria were the presence of keratoconus, poor or unsatisfactory corrected distance visual acuity (CDVA) with spectacle ( ≤ 20/40), intolerance to contact lens, mean keratometry ≥50 D, corneal thickness in the area of the central 3 mm ≤450 μm, and axial length ≥23.50 mm. Exclusion criteria were an active ocular disease (other than keratoconus) and a history of ocular disorders with a potential impact on the variables under study .All DALK surgeries were performed by the same surgeon (JFA) using Anwar's technique . NeoringBriefly, the more significant steps of the surgical procedure were as follows: a partial trephination of the host corneal was performed with a diameter of 8 mm. The donor graft was cut 0.25 mm larger than the size used for the recipient corneal trephination. A manual layer-by-layer stromal dissection was performed, starting at the trephination edge down to the deepest stromal layer adjacent to DM. A thin stromal layer was left behind . A pre-descemetic pocket was created to 0.25 mm outside the trephination of the recipient cornea. Subsequently, a Neoring of 8.5 mm of diameter was implanted in the pre-descemetic pocket. The ring was provisionally fixated to the recipient cornea with two 8-0 silk sutures at 12 and 6 o'clock. Then, the donor button free from DM and endothelium was transferred to the recipient bed and sutured to the host corneal with four interrupted 10-0 nylon sutures (from 90% of the donor button depth and going the suture needle over Neoring to ensure that it is placed at pre-descemetic level). Finally, the silk sutures for the ring fixation were removed, and the 16 interrupted 10-0 nylon sutures were completed.Post-operatively, selective suture removal started at 6 months after DALK, and it was completed at 15 months. The post-operative treatment consisted of a regimen of 1% dexamethasone and ciprofloxacin 0.3% drops four times a day for 1 week. Antibiotic drops were then discontinued, and dexamethasone was progressively tapered down over the next 3 months. Subsequently, we maintained dexamethasone in a regimen of one drop per day for a year. Furthermore, Plasma Rich in Growth Factors (PRGF) eye drops were added topically four times daily for at least 3 months.Post-operative follow-up visits were scheduled 1-, 7-days, and 1 month after surgery and then every 3 months until 24 months . The posP < 0.05 was considered statistically significant. Data are reported as the mean ± SD.Statistical analysis was carried out using Statistical Product and Service Solutions (SPSS) software for Windows . Pre-operative and post-operative data were compared using the Friedman test. A The study included 10 eyes of 10 patients with a mean age of 36.90 ± 2.28 years (range 34–40 years). All patients completed the follow-up period of 2 years and attended all the follow-up visits. P = 0.0001). At the last visit, only four eyes had a spherical refractive error ≥4.00 D. In these cases, the axial length was longer than 25 mm (P < 0.001). At the last visit, no eyes had a post-operative refractive cylinder ≥5 D, and in five eyes (50%), it was ≤2.50 D. Both, the maximum and minimum K reading values, declined significantly after surgery. At the last visit, the mean keratometry was 45.64 ± 1.94 D (ranging from 43.47 to 49.57 D).The spherical component of the refraction dropped from a pre-operative −7.80 ± 3.79 D to a 24-month post-operative value of −3.39 ± 2.58 D . The mean CCT increased from 389.4 ± 51.58 μm pre-operatively to 541.7 ± 34.6 μm at 6 months post-operatively (P = 0.0006), and then it remains unchanged over the follow-up (566.56 ± 30.65 μm at the last visit).There were no significant changes in the mean ECD at any timepoint  was 45.1 ± 24.02 μm. The thickness of the complex host + Neoring was 740.6 ± 35.6 μm, and in all cases, this thickness was thicker than Tg .Advanced keratoconus is a corneal disease that causes severe visual impairment and is a leading indication for corneal transplantation , 13. NotDALK in keratoconus aims to improve visual quality through the restoration of corneal morphology. The graft-host matching is a key factor to achieve optimal visual and refractive outcomes . The thiThis pilot study evaluated the clinical outcomes of implanting a new PMMA ring (Neoring) in a pre-descemetic pocket in the PD-DALK procedure. PD-DALK for keratoconus may provide comparable CDVA outcomes to D-DALK , 20, 21,The results showed satisfactory visual acuity. At the last visit, the CDVA was 0.82 ± 0.14  (range from 0.7 to 1.0), 100% of the eyes achieved a CDVA of 0.7 , and 50% of the eyes achieved a CDVA of 0.8  or better. Furthermore, it is important to note that three of the five eyes with a CDVA of 0.7  had an axial length longer than 26.5 mm and a posterior staphyloma that reached the macular area. Hence, it seems that likely these cases obtained their maximum potential visual acuity. The safety index ratio was 1.87. Our visual results were better than those previously reported in the more advanced keratoconus. In the study of Feizi et al. , the CDVP < 0.001). The average reported refractive cylinder after DALK for keratoconus may vary among studies between 2.50 and 5.00 D  implantation provides significant changes in the anterior and posterior corneal surface morphology of moderate-advanced keratoconus, becoming both surfaces more regular . AlthougFinally, it is essential to note that the implant added volume to the retained peripheral recipient cornea , decreasIn the current study, no complications occurred during surgery or over the entire follow-up time. The most common and significant intraoperative complication is the DM perforation, being mainly associated with the D-DALK technique , 8, 15. Despite the encouraging results in the prospective study, it should be pointed out that this is a pilot study including 10 cases and it only represented a single-center experience. It is important also to note that all surgeries were carried out by an experienced surgeon. Therefore, further prospective, randomized, and multicenter studies with a larger sample and longer follow-ups are required to deeply address the potential benefits of this procedure. Notedly, Neoring was initially conceived for the PD-DALK technique because it is implanted in a PD pocket. However, it should be noted that femtosecond laser can be safely and effectively used to create cutting patterns  in the hIn conclusion, the findings of this pilot study suggest that Neoring implantation in the PD-DALK procedure might represent a viable option to optimize the post-operative results in moderate-severe keratoconus. Further, long-term follow-up studies, including a larger number of cases, are needed to properly analyze the stability of this surgery and confirm the safety of the procedure.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.The study was approved by the Ethics Committee of the Principado de Asturias . The patients/participants provided their written informed consent to participate in this study.BA-B: conception and design of the study, analysis and interpretation of data, writing the manuscript, and critical revision of the manuscript. CL and LF-V-C: conception and design of the study, analysis and interpretation of data, and critical revision of the manuscript. DM-C and JA: conception and design of the study, analysis and interpretation of data, critical revision of the manuscript, and supervision. All authors read and approved the final manuscript.Fernández-Vega Ophthalmological Institute has an unrestricted Grant from AJL Ophthalmic.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody responses remain poorly understood and the clinical utility of serological testing is still unclear.Ct) values of confirmed RT-PCR patients.To understand the relationship between the antibody response to SARS-CoV-2 infection and the demographics and cycle threshold  against SARS-CoV-2. The Ct value, with the highest (87.6 %) positivity observed in individuals with Ct values <20. The mean (±sd) Ct values for IgG positives and negatives were 23.34 (±6.09) and 26.72 (±7.031), respectively. No significant difference was found for demographic characteristics such as age and sex and symptoms and antibody response. The current study is the first of its kind wherein we have assessed the correlation of the RT-PCR Ct with the development of IgG against SARS-CoV-2.IgG positivity was found to be 80.2 % . Positivity increased with a decrease in the Ct values might not have any relation with the development of symptoms, they are associated with the antibody response among SARS-CoV-2-infected individuals.Although  An outbreak of pneumonia was reported in Wuhan, Hubei Province, PR China in late December 2019 . The USAThere is a scarcity of information on the antibody response to SARS-CoV-2 infection . SARS-CoCt) value, gender and age.The present study carried out semi-quantitative SARS-CoV-2 IgG antibody estimation to understand the body’s antibody response in correlation with the severity of SARS-CoV-2 symptoms, cycle threshold  cases were defined as those that tested positive for SARS-CoV-2 RNA using RT-PCR testing of combined nasopharyngeal and throat swab (NT) samples. Patients who presented with one or more symptoms, such as fever, breathlessness, cough, fatigue, muscle pain, clogged nasal cavity, sore throat, diarrhoea, loss of taste (anosmia) and loss of smell (ageusia), during the time of RT-PCR testing were considered symptomatic. This study was approved by the Ethics Committee of ICMR-Regional Medical Research Centre, Bhubaneswar. We obtained informed consent from all participants.A subset of 384 individuals were included in the study to evaluate SARS-CoV-2 IgG between 4 and 6 weeks after being confirmed positive for SARS-CoV-2 by real-time reverse transcription-polymerase chain reaction (RT-PCR) from the month of August to October 2020. The Semi-quantitative SARS-CoV-2 IgG testing was performed using the ARCHITECT i2000SR platform, which uses chemiluminescent microparticle immunoassay (CMIA) technology for the detection of IgG antibodies against the nucleocapsid protein of SARS-CoV-2 from human serum. The cutoff for antibody response was 1.4 index, above which the sample was considered positive.Ct values. A linear treadline was used to show the corelation. Qualitative data were described using frequencies and percentages and analysed using the chi-square test. Quantitative data were described using mean and standard deviation (sd) and analysed using an independent sample t-test. A P-value of less than 0.05 was considered to be statistically significant.Data were entered using MS Excel and descriptive statistical analysis was performed using SPSS software . Scatterplots were used to demostrate the relationship between the antibody titre and P=0.437) (n=334 (86.9 %)] as opposed to females [n=50 (13.1 %)]. Males had a greater chance of producing antibodies than females after a SARS-CoV-2 infection, but the difference was found to be statistically insignificant (P=0.237) (P=0.836). The mean Ct values of symptomatic and symptomatic patients were 23.48±6.070 and 24.16±6.521, respectively. There was no statistical difference for IgG response between symptomatic and asymptomatic patients (P=0.754) (sd) Ct value of the IgG Ab positives was 23.34 (±6.09) and in IgG negatives it was 26.72 (±7.031). The difference in the mean values was found to be statistically significant (P<0.001). The percentage of IgG Ab positives increased with a decrease in the Ct value, which was found to be statistically significant (P<0.001)  of the samples were found to be positive for antibodies against SARS-CoV-2. The median time of the sample collection was 34 days after confirmatory RT-PCR testing. The mean age of the IgG-positive and -negative individuals was 36.94±11.29 and 36.09±10.18, respectively. IgG positivity was found to be highest (88.3 %) in persons aged ≥60 years. No significant difference for antibody response was detected in different age groups (P=0.437) . The samP=0.237) . There wP=0.754) . The meaP<0.001) . The antP<0.001) .In this second phase of the COVID-19 pandemic, sero-testing has emerged as a very useful platform to track down the susceptible population. This method is fast and is considered to be complementary to the gold standard RT-PCR test. Most studies have found a surprisingly lower IgG prevalence (≤90 %) among recovered patients, although a small part of the literature has suggested a higher percentage for the same . This anCt value and IgG antibody titre. Antibody titre was found to be directly proportional to a lower Ct value . Hence, it can be said that higher viral load might lead to the development of a stronger immune response in a SARS-CoV-2-infected individual. Although the kind of immunity exerted by IgG is not yet properly understood, some level of immunity is definitely conferred by IgG, as found in this study. Similar to earlier studies [Ct value and the development of symptoms. One of the earlier studies found that antibody titre cannot be correlated with SARS-CoV-2 disease severity, which can also be corroborated by these data [In this study, the antibody response was found to be 80.2 % among COVID-19-positive individuals, which had been reported by most of the literature . Howeverese data .Ct value by itself cannot be interpreted directly as viral load [Ct can be used as being indicative of viral load in an infected individual.Without the use of a standard curve using reference materials, the ral load , but CtCt values with a semi-quantitative SARS-CoV-2 IgG assay can provide significant assistance in plasma donor selection.There are additional implications from our study for blood banks wherein donors are screened for antibodies using qualitative antibody tests for convalescent plasma to treat COVID-19 patients. To support a previous diagnosis of SARS-CoV-2, these facilities have often relied on self-reporting about patient history and onset of symptoms. The correlation of Ct with the development of IgG against SARS-CoV-2. The Ct value might not have any relation with the severity of the disease, but is associated with the antibody response in SARS-CoV-2-infected persons. However, further long-term studies of longitudinal follow-up of a cohort will help in improving our understanding and forming definitive conclusions.The current study is the first of its kind wherein we have assessed the correlation of RT-PCR


Tenofovir disoproxil fumarate (TDF) is included in first-line antiretroviral treatment (ART) for adolescents living with HIV (ALWH). Associated toxicities remain a concern.We evaluated bone and renal safety outcomes in virologically suppressed South African ALWH after switching to TDF.t-tests) and stratified by sex.We recruited virally suppressed (< 100 copies/mL) adolescents, aged 15–20 years, who switched from an abacavir (ABC)-based to a TDF-based efavirenz regimen. Bone mass and renal function were assessed at Week 0 and at Week 24 after the switch to TDF using dual-energy X-ray absorptiometry (DXA) and serum renal markers. A change in the lumbar spine (LS) and the whole-body less head (WBLH) bone mineral density (BMD) Z-scores and the estimated glomerular filtration rate (eGFR) between the two measures were compared (paired P < 0.0001. The mean change (standard deviation) in the LS-Z-score was –0.03 (0.25) and in the WBLH-Z-score was 0.02 (0.24). A decrease in the eGFR from 132.2 to 120.4 was observed (P = 0.0003); however, the levels remained clinically acceptable.Fifty participants , with a median duration of prior ART of 11.4 years, were enrolled. Among 47 participants with 24-week DXA results, 15 (32%) had either no change or a decreased LS-BMD after the switch, with a mean change of –1.6%. Overall, more female participants experienced this outcome: 58% versus 4%, South African ALWH switching from abacavir to TDF-based ART experienced statistically significant decreases in eGFR but not in LS and WBLH BMD. Female ALWH were more likely to experience a decrease in LS-BMD and may require closer monitoring. Some,13 but not all,17 studies in children report a decline in BMD. Tenofovir alafenamide (TAF), currently unregistered in South Africa (SA), may offer a bone-friendly alternative. Few studies of bone and renal outcomes among TDF-treated children and adolescents exist, especially in sub-Saharan Africa (SSA), where comorbid conditions such as malnutrition, acute and chronic infectious diseases and other adverse environmental conditions are prevalent. The overall objective of this study is to evaluate bone and renal safety after a switch from abacavir (ABC) + 3TC + EFV to the fixed-dose combination (FDC) of TDF + FTC + EFV in clinically stable and virologically suppressed South African ALWH.Low bone mineral density (BMD) and increased fracture risk have been reported in persons living with HIV (PLWH), particularly older men and postmenopausal women.This was a prospective longitudinal study to assess the bone and renal effects of a switch to a TDF-containing ARV regimen in virally controlled, ARV-experienced ALWH.Adolescents living with HIV were recruited from among peers receiving the usual medical care and treatment at Empilweni Clinic of the Rahima Moosa Mother and Child Hospital (RMMCH), Johannesburg, South Africa. Participants were selected through a sample of convenience. We identified all potential participants through the clinic database, conducted file reviews and screened eligible participants at their next scheduled appointment . Informet-tests and 50 subjects. The participants who met the enrolment criteria were between 15 and 20 years of age, had an HIV-1 viral load (VL) of < 100 copies/mL within a 3-month period prior to the initial enrolment visit, weighed ≥ 40 kg and were Tanner stages 4 or 5. An HIV-1 VL of < 100 copies/mL was chosen, as this is the usual clinic cut-off required to switch from an ABC-based to a TDF-based FDC regimen, namely TDF (300 mg) + FTC (200 mg) + EFV (600 mg). Additional criteria included an estimated glomerular filtration rate (eGFR) of ≥ 80 mL/min as per South African national guidelines.18 The exclusion criteria are outlined in Fifty adolescents were enrolled onto our study. Preliminary data from retrospective dual-energy X-ray absorptiometry (DXA) scans performed at the RMMCH on 25 ALWH indicated a 0.32 change (standard deviation [s.d.]: 0.9) in bone accrual over 12 months. Based on this, and assuming an s.d. of 0.9, 80% power and an alpha of 5%, we expected to detect an effect size of 0.36 with paired 19 Therefore, we chose 24 weeks as our primary end point. Week 0 visits were performed between 29 November 2017 and 24 October 2018, and Week 24 visits were performed between 25 May 2018 and 17 April 2019.The study consisted of two visits: Week 0 and Week 24. At Week 0, participants were switched from ABC + 3TC + EFV to the FDC of TDF + FTC + EFV. From other studies, it is evident that the majority of the change in BMD occurs within the first 24 weeks after initiation of TDF.20 A CD4 count was performed if there was no clinic result in the preceding three months. The BMI-for-age and height-for-age Z-scores were assessed using the WHO’s standard growth charts.21At the Week 0 visit, participants were given a questionnaire to complete. A file review provided demographic information , anthropometric measures , a detailed ARV history, vitamin intake, lifestyle choices, a fracture history and the diagnosis of comorbid conditions (current or treated). Participants used a self-assessment tool to define their Tanner stage.Additional study assessments were performed at both visits: DXA of the whole-body less-head (WBLH) and the lumbar spine (LS) for BMD and bone mineral content (BMC) on a single Hologic densitometer with Apex software version 5.6.05 . The bone area-1 size is the scanned area of the bone. The BMD is calculated by dividing the BMC by the bone area-1 size. The precision for BMD and BMC was < 1% for spine and < 2.5% for whole-body phantoms. The BMD and BMC measures are expressed as absolute values and as Z-scores from normative data provided by the Hologic database. Low BMD was defined as a Z-score of ≤ –2.0 on either WBLH or LS. Poor bone outcomes at Week 24 were demonstrated by either no change or a decrease in the BMD Z-score. No change in the BMD Z-score was included in the assessment, as an increase in BMD Z-score is expected in this age group.18 The HIV VL was repeated at Week 24. An undetectable VL was defined as < 50 copies/mL.Blood and urine specimens were collected to check the bone turnover markers and renal function. The bone turnover markers included procollagen type 1 amino-terminal propeptide (P1NP) and the C-terminal telopeptide of type 1 collagen (CTx), which were collected at both study visits and run at one time point to avoid batch effects. The renal markers were serum creatinine, serum urea nitrogen, serum phosphate, serum albumin, urine β-2-microglobulin (β2MG), urine microalbumin, urine phosphate and urine creatinine. These tests were all run in real time. The urine protein:creatinine ratio, urine β2MG creatinine ratio and fractional excretion of phosphate were calculated from these values. The eGFR was calculated using the revised Schwartz equation (2009). According to the South African guidelines at the time of this study, adolescents could be switched to TDF if the eGFR was ≥ 80 mL/min and had to be taken off TDF if the eGFR was < 50 mL/min.t-tests using means and Wilcoxon’s rank-sum tests using medians were conducted for normally distributed and non-normally distributed measurements, respectively.Descriptive statistics were used to characterise the participants at the time of enrolment. Numeric changes in outcomes from Week 0 to Week 24 were calculated, and Shapiro–Wilk tests were conducted to decide if continuous variables were normally distributed. Student’s P-values were two-tailed, and P-values of < 0.05 were considered statistically significant. In addition, 95% confidence intervals (CIs) were calculated. Finally, SAS version 9.4  was used for all statistical analyses.Fisher’s exact tests were used to compare the proportions of poor bone outcome. Univariable logistic regression was used to determine the possible factors associated with poor bone outcome: demographic characteristics, anthropometric measures, ARV uptake, VL, CD4 and changes of bone turnover markers. All Ethical approval was obtained from the University of the Witwatersrand Human Research Ethics Committee  and the Columbia University Institutional Review Board prior to enrolment of participants.One participant was included based on a point-of-care  HIV VL result of < 40 copies/mL; however, the formal VL done at the laboratory was 128 copies/mL. The results were calculated including and excluding this participant, and no significant differences were observed. The results described below include this participant.All participants had complete Week 0 information. Twenty-four (48%) were male, with a median age of 15.5 years (interquartile range [IQR]: 15.1–16.1). While we included participants aged 15–20 years, most were in the younger age range as adolescents in the clinic are switched to the TDF + FTC + EFV FDC as soon as they are eligible. All participants were perinatally HIV infected, with a median duration of ARV use of 11.4 years (IQR: 7.1–13.0). Six (12%) had a prior AIDS-defining illness. The median CD4 count was 728 cells/µL (IQR: 543–1017), and 39 (78%) had an undetectable VL at the Week 0 visit. Nine (18%) had a height-for-age Z-score of ≤ –2 and on BMI, three (6%) were classified as thin and five (10%) as overweight. No participant reported a family history of hip fractures, although six (12%) reported a previous trauma-related fracture .Several follow-up DXA results were excluded because of technical challenges. Consequently, only 47 (94%) participants had 24-week DXA-LS results and 46 (92%) had 24-week DXA-WBLH results. The DXA data were analysed for participants who had at least one DXA result at both time points. At Week 0, the prevalence of low BMD was 13 (28%) for LS and 12 (26%) for WBLH. The median LS and WBLH-Z-scores were –1.15 (IQR: –2.3 to –0.3) and –1.05 (IQR: –2.0 to –0.3), respectively. The mean changes (s.d.) in the LS and WBLH-Z-scores were –0.03 (0.25) and 0.02 (0.24), respectively. No participant had a decrease in the LS-Z-score from > –2.0 to ≤ –2.0, but one participant had this outcome for the WBLH-Z-score.P < 0.0001; 95% CI: 3.5–267.6). At Week 24, 14 (30%) participants demonstrated decreases in WBLH BMD, with a mean change of –1.1%. However, there was no significant difference in reduction of WBLH BMD between female and male participants. There was no difference in the age of those with decreased or unchanged BMD and those with increased BMD. There were no significant associations between poor bone outcomes and the Week 0 weight, Week 0 CD4 count, nadir CD4 count, eGFR or urine protein/creatinine ratio  and decreases in eGFR from 132.2 mL/min to 120.4 mL/min (P = 0.0003). The final levels, however, remained within clinically acceptable limits, and no participants had to be taken off TDF. Although adherence to medication was not specifically addressed, an HIV-1 VL of < 100 indicates good adherence. There was no significant change in HIV-1 VL over the 24 weeks (data not shown).Two participants’ 24-week urine tests were uninterpretable because of menstruation. There were no significant changes in the fractional excretion of phosphate and the protein:creatinine ratio . StatistSouth African ALWH on an EFV-based ART regimen who switched to TDF from other nucleoside reverse transcriptase inhibitors (NRTIs) experienced increases in LS and WBLH BMD over a 6-month period overall, consistent with a preserved trajectory of bone accrual. However, one-third of study participants had poor bone outcomes, with female participants significantly affected compared to male counterparts with regard to the LS-BMD.There was also a small but statistically significant decrease in the eGFR in both female and male participants, of uncertain clinical significance.23 The prevalence of low BMD in our study is similar to that of other middle-income countries, including Brazil (16.7% – 32.4%)25 and Thailand (24%).26 Multiple factors appear to be involved, including the effects of HIV-1 viral proteins, inflammatory cytokines and ART on bone cells and bone turnover.30 Exposure to these factors during childhood and adolescence, which are critical periods for skeletal growth, may impair the attainment of peak bone mass.31 This may have negative effects on adult bone mass and cortical and trabecular bone microarchitecture, which are major determinants of bone strength and fracture risk.31Low BMD is a well-described finding in children and ALWH, with a higher prevalence in middle-income countries compared to resource-rich countries.32 Adolescents have an increased rate of bone accrual during puberty, which occurs earlier in girls. In boys this occurs between 14 years and 17 years and in girls between 11 years and 14 years; healthy female individuals have a decrease in the rate of bone accumulation from the age of 16 years.34 The median age of our participants was 15.5 years. More female than male participants experienced a decrease in LS-BMD, which may be associated with a decrease in bone accrual in girls at this age.Ninety per cent of peak bone mass is attained by the age of 18 years.14 and 10 years35 of follow-up. Aurpibul et al. found a significant decrease in BMD over 96 weeks in virologically suppressed children and adolescents who were on a non-nucleoside reverse transcriptase inhibitor-based regimen prior to switching to TDF + 3TC + EFV.13 Some of the difference in findings between the studies may be attributed to an improvement in BMD that may have occurred with a concurrent switch off protease inhibitors, cancelling the negative effects of TDF initiation.36 In this study, by maintaining EFV, and just switching the NRTI component, we were able to isolate the effects of switching to a single agent. However, these results may not be generalisable if the TDF switch occurs with a protease inhibitor as the backbone ARV.The independent effects of specific ARVs on BMD, either at ART initiation or upon switching, are sometimes difficult to ascertain because ARVs are given as combinations. An observed improvement in BMD may be the result of either a switch off an agent with negative effects on bone or the addition of an agent with a safer bone profile. Two studies that looked at a switch from stavudine (D4T) + 3TC + lopinavir/ritonavir to TDF + 3TC/FTC + EFV in virologically suppressed children and adolescents found no significant changes in BMD over 60 monthsOur study demonstrated that TDF initiation resulted in an overall increase in BMD among adolescents, which is the expected trajectory during growth; however, the effect differed between individuals and by sex, with a greater proportion of female participants experiencing net bone loss. Weight at the time of switch was not predictive of change in BMD, nor bone turnover marker levels. We also did not find an association between higher fractional excretion of phosphate or change in eGFR and bone loss. Our findings suggest that a switch to TDF may have negative consequences for specific individuals, especially girls; however, we cannot predict which individuals will experience greater bone loss based on clinical or laboratory parameters.37 Adult studies have demonstrated increased levels of CTx and P1NP in patients initiating ART irrespective of the regimen, but higher on TDF-containing ART.44 Levels increased in the first 24–48 weeks after ART initiation, and then decreased.44 Few studies have looked at bone biomarkers in children and adolescents. In Thai adolescents, TDF use was positively associated with CTx but negatively with P1NP when compared to non-TDF.45 This indicates abnormal rather than increased bone metabolism; this was not associated with low BMD in this study.45 There was no significant change in CTx or P1NP at 24 weeks after the switch to TDF, probably because our participants were ARV experienced and well suppressed at the time of the switch.Tenofovir disoproxil fumarate-associated BMD loss is not well understood; however, one mechanism may be abnormal bone metabolism.46 There is little data on the renal effects of TDF on children and adolescents in SSA.Another mechanism of TDF-associated bone loss is chronic phosphaturia and hypophosphataemia resulting from renal tubular dysfunction.47 Proteinuria and a lower eGFR were associated with concomitant protease inhibitor (PI) use.47 It is difficult to compare studies, as participants in the Zimbabwean study were younger with a shorter duration of ART, and VL was not evaluated because of cost restraints. We found no evidence of hypophosphataemia, phosphaturia, proteinuria or renal impairment 24 weeks after the switch to TDF-containing ART. These findings are similar to other studies that have looked at virally suppressed children and adolescents switching to a TDF-containing regimen49; however, in a study extended to 132 weeks, 46% of patients developed mild or moderate hypophosphataemia.15 While most cases were mild and not clinically relevant,15 longer-term monitoring may be needed as this may contribute to low BMD over time.46A cross-sectional study of Zimbabwean children and adolescents < 18 years and on TDF found hypophosphataemia, proteinuria and a decreased eGFR of < 90 mL/min per 1.73 m² in 11.1%, 32.8% and 35.9%, respectively.50 Our study was conducted when all eligible adolescents were switched to TDF + FTC + EFV from age 15 years, and it is therefore restricted to participants remaining on EFV. Integrase strand transfer inhibitor-based regimens with either TAF or ABC had minimal effects on BMD51; however, these regimens are not currently available in SA, either as FDCs or at all. While we advocate for a bone-sparing regimen in the adolescent population, TDF is likely to be used in SA and other low- and middle-income countries for the foreseeable future. Further research on the long-term effects of TDF in this population and younger adolescents from age 10 years is needed.Currently in SA, adolescents from age 10 years who are suppressed on AZT/ABC/D4T + 3TC/FTC + EFV are being switched to TDF + 3TC/FTC with dolutegravir or high-dose EFV as an FDC after appropriate counselling.One limitation of our study is the lack of longitudinal data in healthy South African controls of similar age to determine whether the rate of bone accrual is similar to uninfected controls. Similarly, we lack data on the rate of change of BMD and BMC in ALWH who have not been switched to TDF-containing regimens.52 However, other studies have shown decreasing BMD at 48 weeks and then a stabilisation of the BMD13; therefore, no significant changes in BMD at 24 weeks are promising. The Z-scores were not height adjusted, which may have overestimated the prevalence of low BMD at the time of the switch to TDF45; however, this does not affect our comparison between the two time points. Information on the participants’ diet, calcium intake and exercise was not included in this study, and so the effect of these factors on BMD changes could not be assessed.Additionally, this was a small study of 50 participants, and because of technical difficulties, several patients did not have 24-week DXA results, which resulted in wide confidence intervals related to sex differences for LS-BMD. The significance of our results must be interpreted with this in mind. Furthermore, we have only 24 weeks of follow-up, which did not allow us to determine the long-term effects of TDF in this population, and the time interval between the DXA scans may have been too short to show changes in these older adolescents.In conclusion, we demonstrate that switching to TDF with an EFV-based regimen in virally suppressed ALWH results in overall gains in BMD commensurate with bone acquisition; however, more female than male participants experienced a 6-month decline in BMD and may require monitoring with DXA scans after the switch. Statistically significant reductions in eGFR were demonstrated 24 weeks after the switch to TDF, although eGFR was still clinically acceptable and well above the threshold at which guidelines recommend TDF withdrawal. Future research into the long-term effects of TDF on bone strength and renal function in this population, as well as in younger adolescents, is warranted.